1. Field of the Invention
The present invention relates generally to molecular assemblies, and more particularly to an assay for the detection of GPCR molecular assemblies and bead-based detection of ligand-GPCR complexes.
2. Description of the Prior Art
G protein coupled receptors (GPCRs) interact with extracellular stimuli, such as photons, hormones, neurotransmitters, and odorants, see Gilman A G (1995), Nobel Lecture, G Proteins and Regulation of Adenylyl Cyclase, Biosci Rep, 15, pp 65–97, the entire contents and disclosure of which is hereby incorporated by reference. These stimuli cause coformational changes in the receptor leading to binding of intracellular G protein heterotrimers, each with one copy of a guanyl nucleotide binding α subunit, and a βγ dimer, see Neer E J (1995), Heterotrimeric G Proteins: Organizers of Transmembrane Signals, Cell, 80, pp 249–257, the entire contents and disclosure of which is hereby incorporated by reference. After stimulation, the α subunit binds GTP, which promotes dissociation of the α subunit from the βγ dimer, exposing new surfaces to cytoplasmic effectors, such as adenylyl cyclase and phospholipase C. The human genome contains ˜600 GPCR genes, 27 α, 5 β, and 13 γ, see Venter et al. (2001), The Sequence of the Human Genome, Science, 291, pp 1304–1351, the entire contents and disclosure of which is hereby incorporated by reference, with smaller numbers of these G proteins (17, 5 and 12, respectively) found to date. With such large numbers, determining how productively any given GPCR couples to a particular αβγ heterotrimer is daunting (1,020 αβγ combinations alone). The assembly of a high agonist-affinity complex is a good criterion of productive partners, see Gilman A G (1987), G Proteins: Transducers of Receptor-Generated Signals, Ann Rev Biochem, 56, pp 615–649, the entire contents and disclosure of which is hereby incorporated by reference.
The formyl peptide receptor (FPR) responds to the presence of N-formyl methionine-containing peptides resulting from bacterial and mitochondrial protein synthesis, as well as other hydrophobic peptides, see Gao et al. (1994), A High Potency Nonformylated Peptide Agonist for the Phagocyte N-Formylpeptide Chemotactic Receptor, J Exp Med, 180, pp 2191–2197, the entire contents and disclosure of which is hereby incorporated by reference. This receptor has served as a model for signal transduction in phagocytic cells and for inflammatory and autoimmune diseases, see Prossnitz E R and Ye R D (1997), The N-Formyl Peptide Receptor: a Model for the Study of Chemoattractant Receptor Structure and Function, Pharacol Ther, 74, pp 73–102, the entire contents and disclosure of which is hereby incorporated by reference. The receptor has been cloned and overexpressed in tissue culture cells, solubilized, and assembled with a formyl peptide ligand and G protein to form a high agonist-affinity ternary complex in solution, see Bennett et al. (2001), Real-Time Analysis of G Protein-Coupled Receptor Reconstitution in a Solubilized System, J Biol Chem, 276, pp 22453–22460, the entire contents and disclosure of which is hereby incorporated by reference.
The soluble receptor reconstitutes with ligand, G proteins, and arrestin in a manner that is sensitive to receptor phosphorylation and mutations in both the receptor and G proteins. The assembly may be measured in real-time with fluorescent ligands, and the assemblies are consistent with cellular co-localizations observed by fluorescence confocal microscopy, see Bennett et al. (2001), Real-Time Analysis of G Protein-Coupled Receptor Reconstitution in a Soubilized System, J Biol Chem, 276, pp 22453–22460; Bennett et al. (2001), Partial Phosphorylation of the N-Formyl Peptide Receptor Inhibits G Protein Association Independent of Arrestin Binding, J Biol Chem, 276, pp 49195–49203; and Key et al. (2001), Regulation of Formyl Peptide Receptor Agonist Affinity by Reconstitution with Arrestins and Heterotrimeric G Proteins, J Biol Chem, 276, pp 49204–49212, the entire contents and disclosures of which are hereby incorporated by reference.
While ternary complex assemblies have been the subject of experimental investigation and mathematical modeling over several decades, see Kent et al. (1980), A Quantitative Analysis of Beta-Adrenergic Receptor Interactions: Resolution of High and Low Affinity States of the Receptor by Computer Modeling of Ligand Binding Data, Mol Pharmacol, 17, pp 14–23, the entire contents and disclosure of which is hereby incorporated by reference, the tools to examine the affinities and kinetics of individual steps in complex formation, disassembly, activation, and termination have only been accessible in a limited way, see Christopoulos A and Kenakin T (2002), G Protein-Coupled Receptor Allosterism and Complexing, Pharmacol Rev, 54, pp 323–374, the entire contents and disclosure of which is hereby incorporated by reference. For rhodopsin, it has been possible to measure complex assembly and disassembly through the spectroscopic signature of the metarhodopsin II-transducin complex, see Mitchell et al. (2001), Optimization of Receptor-G Protein Coupling by Bilayer Lipid Composition I: Kinetics of Rhodopsin-Transducin Binding, J Biol Chem, 276, pp 42801–42806, the entire contents and disclosure of which is hereby incorporated by reference. GPCRs also activate transmembrane channels in the subsecond time frame, see Mark et al. (2000), G Protein Modulation of Recombinant P/Q-Type Calcium Channels by Regulators of G Protein Signaling Proteins, J Physiol, 528, Pt. 1, pp 65–77, the entire contents and disclosure of which is hereby incorporated by reference, where ternary complex dynamics can be inferred from measurements of ion currents. Such measurements have given a Gt (transducin) activation rate of ˜120 s−1, see Leskov et al. (2000), The Gain of Rod Phototransduction: Reconciliation of Biochemical and Electrophysiological Measurements, Neuron, 27, pp 525–537, the entire contents and disclosure of which is hereby incorporated by reference, probably unique to the visual transduction system, and a Gq (a heterotrimeric G protein in which the α subunit is the q subtype, αq) activation rate of 2 s−1, Muldiopadhyay S and Ross E M (1999), Rapid GTP Binding and Hydrolysis by G(q) Promoted by Receptor and GTPase-Activating Proteins, Proc Natl Acad Sci USA, 96, pp 9539–9544, the entire contents and disclosure of which is hereby incorporated by reference. Both surface plasmon resonance, see Rebois (2002), Elucidating Kinetic and Thermodynamic Constants for Interaction of G Protein Subunits and Receptors by Surface Plasmon Resonance Spectoscopy, Methods in Enzymology (Iyengar Ra and Hildebrandt J D, eds), Academic Press, New York, pp 15–42, the entire contents and disclosure of which is hereby incorporated by reference, and flow cytometry, see Nolan J P and Sklar L A (1998), The Emergence of Flow Cytometry for Sensitive, Real-Time Measurements of Molecular Interactions, Nat Biotechnol, 16, pp 633–638, the entire contents and disclosure of which is hereby incorporated by reference, could be general tools for measuring individual rate constants.
Therefore, appropriate tools in these areas have been limited and, in many situations, unsatisfactory. Thus, there is a need for a homogeneous, small volume bead-based approach compatible with high throughput flow cytometry, which would allow evaluation of G protein coupled receptor molecular assemblies.